title: High-throughput fluorescence anisotropy screen for inhibitors of the oncogenic mRNA binding protein, IMP-1.
abstract: Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes messenger RNAs (mRNAs), leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung, and colon cancer. Small-molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z' factor = 0.60) FAMA-based high-throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic nonspecific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high-throughput screen was demonstrated by identification of the first small-molecule IMP-1 inhibitor, a lead compound that selectively inhibits proliferation of IMP-1-positive cancer cells with very little or no effect on proliferation of IMP-1-negative cells.
authors.affiliation: 1Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL, USA.
authors.forename: Lily
authors.initials: L
authors.lastname: Mahapatra
authors.forename: Chengjian
authors.initials: C
authors.lastname: Mao
authors.forename: Neal
authors.initials: N
authors.lastname: Andruska
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.forename: David J
authors.initials: DJ
authors.lastname: Shapiro
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D055808:Drug Discovery; D004354:Drug Screening Assays, Antitumor; D005454:Fluorescence Polarization / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
chemical_list: C406144:IGF2BP1 protein, human; D016601:RNA-Binding Proteins; D054852:Small Molecule Libraries
keywords: IMP-1; RNA-binding protein; cancer; fluorescence anisotropy/polarization; inhibitor